Abstract
Several RNA silencing strategies employing antisense or inverted repeat constructs have been applied to Chlamydomonas reinhardtii. Problems inherent to these strategies, like off-target effects by unpredictable generation of siRNAs, were solved previously by constructs allowing for routine expression of specific artificial microRNAs (amiRNAs). Yet missing was a routine tool for inducible amiRNA expression, which to establish was the aim of this work. For this, we equipped a recently developed amiRNA expression vector with the NIT1 promoter, which is repressed by ammonium and activated by nitrate. We tested this conditional amiRNA vector with heat shock factor 1 (HSF1) as target. HSF1 transcripts in transformants were already reduced ~2 h after transfer from ammonium to nitrate-containing medium. In contrast, HSF1 protein levels declined only ~8 h after the shift and were strongly reduced after 24 h, suggesting that HSF1 is a stable protein and diluted out by growth. HSF1 levels recovered partly when transformant cells were shifted back to ammonium for 72 h. Transformants developed thermosensitivity only on nitrate and thermosensitivity correlated with strong reduction in HSF1 levels, hence supporting our earlier conclusion that HSF1 is a key regulator for thermotolerance in Chlamydomonas.
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Acknowledgments
We thank Steve Miller for critically reading the manuscript, Attila Molnar for construct pChlamiRNA2 and helpful advice, and Olivier Vallon for the antiserum against CF1β. This work was supported by the Max Planck Society and grants from the Deutsche Forschungsgemeinschaft (Schr 617/5-1) and the Bundesministerium für Bildung und Forschung (Systems Biology Initiative FORSYS, project GoFORSYS).
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Communicated by A. Grossman.
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Schmollinger, S., Strenkert, D. & Schroda, M. An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance. Curr Genet 56, 383–389 (2010). https://doi.org/10.1007/s00294-010-0304-4
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DOI: https://doi.org/10.1007/s00294-010-0304-4